p53 is a tumor suppresser protein that plays a central role in protection against development of cancer. It guards cellular integrity and prevents the propagation of permanently damaged clones of cells by the induction of growth arrest or apoptosis. At the molecular level, p53 is a transcription factor that can activate a panel of genes implicated in the regulation of cell cycle and apoptosis. p53 is a potent cell cycle inhibitor which is tightly regulated by MDM2 at the cellular level. MDM2 and p53 form a feedback control loop. MDM2 can bind p53 and inhibit its ability to transactivate p53-regulated genes. In addition, MDM2 mediates the ubiquitin-dependent degradation of p53. p53 can activate the expression of the MDM2 gene, thus raising the cellular level of MDM2 protein. This feedback control loop insures that both MDM2 and p53 are kept at a low level in normal proliferating cells. MDM2 is also a cofactor for E2F, which plays a central role in cell cycle regulation.
The ratio of MDM2 to p53 (E2F) is dysregulated in many cancers. Frequently occurring molecular defects in the p16INK4/p19ARF locus, for instance, have been shown to affect MDM2 protein degradation. Inhibition of MDM2-p53 interaction in tumor cells with wild-type p53 should lead to accumulation of p53, cell cycle arrest and/or apoptosis. MDM2 antagonists, therefore, can offer a novel approach to cancer therapy as single agents or in combination.with a broad spectrum of other antitumor therapies. The feasibility of this strategy has been shown by the use of different macromolecular tools for inhibition of MDM2-p53 interaction (e.g. antibodies, antisense oligonucleotides, peptides). MDM2 also binds E2F through a conserved binding region as p53 and activates E2F-dependent transcription of cyclin A, suggesting that MDM2 antagonists might have effects in p53 mutant cells.
Wells et al. J. Org. Chem., 1972, 37, 2158-2161, report synthesis of imidazolines. Hunter et al. Can. J. Chem., 1972, Vol. 50, 669-77, report the preparation of amarine and isoamarine compounds which had previously been studied for chemiluminescence (McCapra et al. Photochem. and Photobiol. 1965, 4, 1111-1121). Zupanc et al. Bull. Soc. Chem. and Tech. (Yugoslavia) 1980-81, 27/28, 71-80, report the use of triaryl imidazolines as starting materials in the preparation of EDTA derivatives. EP 363 061 to Matsumoto reports imidazoline derivaties useful as immunomodulators. The compounds were indicated to have low toxicity. Treatment and/or prevention of rheumatoid arthritis, multiple sclerosis, systemic lupus, erythemathodes, and rheumatic fever were implicated. WO 00/78725 to Choueiry et al. report a method for making substituted amidine compounds, and indicate that imidazoline-type compounds may be useful in the treatment of diabetes or related diseases involving impaired glucose disposal.
The present invention provides at least one compound selected from a compound of formula I 
and the pharmaceutically acceptable salts and esters thereof, wherein
Z1, Z2 and Z3 are each independently selected from lower alkoxy, xe2x80x94CH2OCH3, and xe2x80x94CH2OCH2CH3,
or one of Z1, Z2 or Z3 is xe2x80x94H and the other two are each independently selected from lower alkyl, lower alkoxy, xe2x80x94Cl, xe2x80x94Br, xe2x80x94F, xe2x80x94CF3, xe2x80x94CH2OCH3, xe2x80x94CH2OCH2CH3, xe2x80x94OCH2CH2R1, xe2x80x94CH2-morpholino, xe2x80x94OR2, xe2x80x94CH2R2, xe2x80x94OCH2CF3, xe2x80x94OCH(CH3)CH2OH and xe2x80x94COOQ,
wherein Q is selected from xe2x80x94H and lower alkyl,
or one of Z1, Z2 or Z3 is xe2x80x94H and the other two taken together with the two carbon atoms and the bonds between them from the benzene ring to which they are substituted form a ring selected from 5- and 6-membered unsaturated rings, and 5- and 6-membered saturated rings that contain at least one hetero atom selected from S, N, and O,
wherein R1 is selected from xe2x80x94F, xe2x80x94OCH3, xe2x80x94N(CH3)2, and unsaturated 5-membered rings containing at least one hetero atom wherein the hetero atom is selected from S, N and O,
wherein R2 is a 3- to 6-membered saturated ring, and
Y1 and Y2 are each independently selected from xe2x80x94Cl, xe2x80x94Br, xe2x80x94NO2, xe2x80x94Cxe2x89xa1N and Cxe2x89xa1CH.
The present invention also provides at least one compound selected from a compound of formula II 
and the pharmaceutically acceptable salts and esters thereof, wherein
Z4 is selected from C1-C2 alkyl, lower alkoxy, xe2x80x94OH, xe2x80x94SCH3, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94COOQ2, xe2x80x94N(CH3)2, xe2x80x94OCH2-phenyl, xe2x80x94Cl, xe2x80x94Br, xe2x80x94F, xe2x80x94OCH2Cxe2x95x90OOQ1, saturated 5- and 6-membered rings containing at least one hetero atom wherein the hetero atom is selected from S, N and O,
wherein Q1 is selected from xe2x80x94H, xe2x80x94NH2, and lower alkyl,
wherein Q2 is selected from xe2x80x94H and lower alkyl,
Y1 and Y2 are independently selected from xe2x80x94Cl, xe2x80x94Br, xe2x80x94NO2 and xe2x80x94CN
with the proviso that where Y1 and Y2 are both xe2x80x94Cl, then Z4 is not xe2x80x94Cl,
with the proviso that where Y1 and Y2 are both xe2x80x94NO2, then Z4 is not xe2x80x94NO2, and
with the proviso that where Y1 and Y2 are both xe2x80x94CN, then Z4 is not xe2x80x94CN.
The present invention provides cis-imidazolines which are small molecule inhibitors of the MDM2-p53 interaction. In cell-free and cell-based assays, compounds of the present invention are shown to inhibit the interaction of MDM2 protein with a p53-like peptide with a potency that is approximately 100 fold greater than a p53-derived peptide. In cell-based assays, these compounds have demonstrated mechanistic activity. Incubation of cancer cells with wild-type p53 has led to accumulation of p53 protein, induction of p53-regulated p21 gene, and cell cycle arrest in G1 and G2 phase. This resulted in potent antiproliferative activity against wild-type p53 cells in vitro. In contrast, these activities were not observed in cancer cells with mutant p53 at comparable compound concentrations. Therefore, the activity of MDM2 antagonists is likely linked to its mechanism of action. These compounds can be potent and selective anticancer agents.
The present invention provides at least one compound selected from a compound of formula I 
and the pharmaceutically acceptable salts and esters thereof, wherein
Z1, Z2 and Z3 are each independently selected from lower alkoxy, xe2x80x94CH2OCH3, and xe2x80x94CH2OCH2CH3,
or one of Z1, Z2 or Z3 is xe2x80x94H and the other two are each independently selected from lower alkyl, lower alkoxy, xe2x80x94Cl, xe2x80x94Br, xe2x80x94F, xe2x80x94CF3, xe2x80x94CH2OCH3, xe2x80x94CH2OCH2CH3, xe2x80x94OCH2CH2R1, xe2x80x94CH2-morpholino, xe2x80x94OR2, xe2x80x94CH2R2, xe2x80x94OCH2CF3, xe2x80x94OCH(CH3)CH2OH and xe2x80x94COOQ,
wherein Q is selected from xe2x80x94H and lower alkyl,
or one of Z1, Z2 or Z3 is xe2x80x94H and the other two taken together with the two carbon atoms and the bonds between them from the benzene ring to which they are substituted form a ring selected from 5- and 6-membered unsaturated rings, and 5- and 6-membered saturated rings that contain at least one hetero atom selected from S, N, and O,
wherein R1 is selected from xe2x80x94F, xe2x80x94OCH3, xe2x80x94N(CH3)2, and unsaturated 5-membered rings containing at least one hetero atom wherein the hetero atom is selected from S, N and O,
wherein R2 is a 3- to 6-membered saturated ring, and
Y1 and Y2 are each independently selected from xe2x80x94Cl, xe2x80x94Br, xe2x80x94NO2 and xe2x80x94Cxe2x89xa1N and xe2x80x94Cxe2x89xa1CH.
The present invention also provides at least one compound selected from a compound of formula II 
and the pharmaceutically acceptable salts and esters thereof, wherein
Z4 is selected from C1-C2 alkyl, lower alkoxy, xe2x80x94OH, xe2x80x94SCH3, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94COOQ2,
xe2x80x94N(CH3)2, xe2x80x94OCH2-phenyl, xe2x80x94Cl, xe2x80x94Br, xe2x80x94F, xe2x80x94OCH2Cxe2x95x90OOQ1, saturated 5- and 6-membered rings containing at least one hetero atom wherein the hetero atom is selected from S, N and O,
wherein Q1 is selected from xe2x80x94H, xe2x80x94NH2, and lower alkyl,
wherein Q2 is selected from xe2x80x94H and lower alkyl,
Y1 and Y2 are independently selected from xe2x80x94Cl, xe2x80x94Br, xe2x80x94NO2, xe2x80x94Cxe2x89xa1N and xe2x80x94Cxe2x89xa1CH.
with the proviso that where Y1 and Y2 are both xe2x80x94Cl, then Z4 is not xe2x80x94Cl,
with the proviso that where Y1 and Y2 are both xe2x80x94NO2, then Z4 is not xe2x80x94NO2, and
with the proviso that where Y1 and Y2 are both xe2x80x94CN, then Z4 is not xe2x80x94CN.
xe2x80x9cEffective amountxe2x80x9d means an amount that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
xe2x80x9cHalogenxe2x80x9d means fluorine, chlorine, bromine or iodine.
xe2x80x9cHetero atomxe2x80x9d means an atom selected from N, O and S.
xe2x80x9cIC50xe2x80x9d refers to the concentration of a particular compound required to inhibit 50% of a specific measured activity. IC50 can be measured, inter alia, as is described subsequently.
xe2x80x9cAlkylxe2x80x9d denotes a straight-chained or branched saturated aliphatic hydrocarbon. xe2x80x9cLower alkylxe2x80x9d groups denote C1-C6 alkyl groups and include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, 2-butyl, pentyl, hexyl, and the like. Generally, lower alkyl is preferably C1-C4 alkyl, and more preferably C1-C3 alkyl.
xe2x80x9cAlkoxyxe2x80x9d denotes xe2x80x94O-alkyl. xe2x80x9cLower alkoxyxe2x80x9d denotes xe2x80x94O-lower alkyl.
xe2x80x9cPharmaceutically acceptable esterxe2x80x9d refers to a conventionally esterified compound of formula I having a carboxyl group, which esters retain the biological effectiveness and properties of the compounds of formula I and are cleaved in vivo (in the organism) to the corresponding active carboxylic acid.
Information concerning esters and the use of esters for the delivery of pharmaceutical compounds is available in Design of Prodrugs. Bundgaard H ed. (Elsevier, 1985). See also, H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 108-109; Krogsgaard-Larsen, et. al., Textbook of Drug Design and Development (2d Ed. 1996) at pp. 152-191.
xe2x80x9cPharmaceutically acceptable saltxe2x80x9d refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide. Chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. See, e.g., H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.
xe2x80x9cPharmaceutically acceptable,xe2x80x9d such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.
xe2x80x9cSubstituted,xe2x80x9d as in substituted alkyl, means that the substitution can occur at one or more positions and, unless otherwise indicated, that the substituents at each substitution site are independently selected from the specified options.
xe2x80x9cTherapeutically effective amountxe2x80x9d means an amount of at least one compound of the present invention, that significantly inhibits proliferation and/or prevents differentiation of a human tumor cell, including human tumor cell lines.
Compounds of the present invention as exemplified advantageously show IC50s from about 0.5 xcexcM to about 300 xcexcM.
The compounds of the present invention are useful in the treatment or control of cell proliferative disorders, in particular oncological disorders. These compounds and formulations containing said compounds may be useful in the treatment or control of solid tumors, such as, for example, breast, colon, lung and prostate tumors.
A therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion.
The compounds of the present invention can be prepared according to the following schemes. The following definitions are provided as applicable to the synthesis schemes:
V1, V2, V3, V4, V5 are each independently selected from the group of:
hydrogen,
xe2x80x94OV6,
xe2x80x94SV7,
xe2x80x94NV8V9,
xe2x80x94CONV8V9,
xe2x80x94COOV10 
halogen,
nitro,
trifluoromethyl,
lower alkyl, which optionally may be substituted by V11, and
cycloalkyl;
V1, V2 together may form part of a heterocycle with one or more hetereoatoms, which optionally may be substituted by V10.
V2, V3 together may form part of a heterocycle with one or more hetereoatoms, which optionally may be substituted by V10.
Y1, Y2 are each independently selected from the group of:
xe2x80x94Cl,
xe2x80x94Br,
nitro,
cyano, and
xe2x80x94Cxe2x89xa1CH;
V6 is selected from the group of:
hydrogen,
lower alkyl, which optionally may be substituted by V11, and
cycloalkyl;
V7 is selected from the group of:
hydrogen, and
lower alkyl;
V8, V9 are each independently selected from the group of:
hydrogen,
lower alkyl,
cycloalkyl; or
V8, V9 together may form part of a hetereocycle with one or more hetereoatoms;
V10 is selected from the group of:
hydrogen,
lower alkyl, and
cycloalkyl;
V11 is selected from the group of:
xe2x80x94CONV8V9 
xe2x80x94NV8V9,
xe2x80x94COOV10,
aryl,
halogen,
lower alkoxy,
morpholinyl, and
5-membered heterocycles;
The cis isomers of formula I are preferred. 
Many benzonitriles of formula 2 are commercially available. They are converted to the imidate salts (3) using HCl gas in ethanol. The rate of the reaction depends on the substituents on the phenyl ring. In cases where V1xe2x89xa0H, it may be necessary to run the reaction under pressure of HCl over a longer period of time. Condensation of the imidates (3) with the 1,2-diamines (4) is carried out in ethanol at 40-100xc2x0 C. in the presence or absence of a base such as triethylamine.
The meso-1,2-diamines of formula 4 (Y1=Y2) are known compounds and prepared according to the literature procedures (see Jennerwein, M. et al. Cancer Res. Clin. Oncol. 1988, 114, 347-58; Vogtle, F.; Goldschmitt, E. Chem. Ber. 1976, 109, 1-40).
If it is desired to prepare the 1,2-diamines of formula 4 wherein Y1xe2x89xa0Y2, modifications to the existing procedures (vide supra) can be made. An equal molar mixture of the benzaldehydes and meso-1,2-bis-(2-hydroxy-phenyl)-ethane-1,2-diamine can be used to afford a mixture of 1,2-diamines (Scheme II). It was then reacted with compound of formula 3 to give a mixture of products. The desired compound (1) can be isolated from the mixture by preparative chromatography techniques. 
The imidate salts (6) can be prepared from the amides of formula 5 using triethyloxonium tetrafluoroborate in methylene chloride (Scheme III), a method known in the art (Weintraub, L.; Oles, S. R.; Kalish, N. J. Org. Chem. 1968, 33, 1679-1681). The imidate salts (6) are then condensed with the diamines (4) in the same manner as described for the imidates (3). 
The compounds of formula 1 can be prepared directly by a condensation reaction of the benzoic acids (7) with the 1,2-diamines (4) (Scheme IV). The yields are low and thus it can be useful in cases where methods of preparation of imidates (3 and 4) have failed (see Hammouda, H. A.; Abd-Allah, S. O.; Sharaf, M. A. F. Egypt. J. Chem. 1987, 30, 239-247). 
Benzonitriles of formula 2 (of Scheme I) may be prepared in accordance with the following methods.
The benzonitriles of formula 9 (V can be any suitable group such as for V1, V2, V3, V4, or V5) can be prepared by alkylation of phenols 8 with V6X (X=Cl, Br, I) using conventional methods (Scheme V). The phenoxide anion is generated by a base such as cesium carbonate or potassium carbonate. The reaction typically runs at reflux temperature of a solvent such as ethanol. V6 can also be introduced using Mitsunobu reaction (see for example, Hughes, D. L. Org. React. 1992, 42, 335-656). 
Aromatic aldehydes 10 (V can be any suitable group such as for V1, V2, V3, V4, or V5) can be converted into benzonitriles using literature procedures (Karmarkar, S. N; Kelkar, S. L.; Wadia, M. S. Synthesis 1985, 510-512; Bergeron, R. J. et al. J. Med. Chem. 1999, 42, 95-108). V6 group can then be introduced using V6X (X=Cl, Br, I) or Mitsunobu reaction to give the benzonitriles 11 (Scheme VI). 
The halides of formula 13 can be prepared by bromination or iodination of phenols (12) (Scheme VII, V can be any suitable group such as for V1, V2, V3, V4, or V5). Reaction conditions such as N-bromosuccinimide/tetrahydrofuran or iodine/thallium acetate can be utilized (see for example, Carreno, M. C.; Garcia Ruano, J. L.; Sanz, G.; Toledo, M. A.; Urbano, A. Synlett 1997, 1241-1242; Cambie, R. C.; Rutledge, P. S.; Smith-Palmer, T.; Woodgate, P. D. J. Chem. Soc., Perkin Trans. 1 1976, 1161-4). V6 group can then be introduced using V6X (X=Cl, Br, I) or Mitsunobu reaction. Methods of converting aromatic halides to the corresponding nitrites are known in the art (see for example, Okano, T.; Iwahara, M.; Kiji, J., Synlett 1998, 243). Cyanation of halides 13 (Xxe2x80x2=Br, I) is accomplished using zinc cyanide with a catalyst such as tetrakis(triphenyl-phosphine)palladium. Solvents such dimethylformamide can be used and the reaction temperature is between 80-110xc2x0 C. 
In Scheme VIII, amination of aromatic halides using HNV8V9 and palladium catalyst can be utilized to provide the benzonitriles of formula 15 (see for example, Harris, M. C.; Geis, O.; Buchwald, S. L. J. Org. Chem. 1999, 64, 6019). 
Various benzonitriles of formula 11 (V can be any suitable group such as V1, V2, V3, V4, or V5) can be prepared by nucleophilic substitution of benzonitriles (16) (Scheme 9). Procedures to affect that transformation are reported in the literature (see for example, X=F: Wells, K. M.; Shi, Y.-J.; Lynch, J. E.; Humphrey, G. R.; Volante, R. P.; Reider, P. J. Tetrahedron Lett. 1996, 37, 6439-6442; X=NO2: Harrison, C. R.; Lett, R. M.; McCann, S. F.; Shapiro, R.; Stevenson, T. M. WO 92/03421, 1992). 
Methyl ester of 5-bromoisophthalic acid can be utilized to prepare a variety of 3,5-disubstituted benzonitriles (18) wherein W=xe2x80x94CH2OV6, xe2x80x94CH2NV8V9, xe2x80x94CH2-(morpholine), xe2x80x94CONV8V9, xe2x80x94COOV10 (Scheme X). Suitable functional group transformations (FGT) can convert the ester moiety to various other groups (e.g. carboxylic acid, amide, alcohol, halide, ether, amine, etc.), as is known in the art. 
To prepare benzonitriles of formula 21 wherein V1, V2, V3, V4, or V5=OV6, sequential alkylation of the diols (19) with suitable V6X (X=Cl, Br, I) are used. The bromides (20) are then converted to the nitrites (21) using zinc cyanide and Pd(0) catalyst (Scheme XI). 
Method of preparation of 3-bromo-5-methoxy-phenyl-methanol from 3-bromo-5-methyl-phenol (see Claudi, F. et al. J. Med. Chem. 2000, 43, 599-608) can be adapted to provide various benzonitriles of formula 24 (Scheme XII). The phenols 22 can be alkylated with V6X (X=Cl, Br, I). Oxidation of methyl group using potassium permanganate provides the carboxylic acid (23). Manipulation of the carboxylic acid moiety leads to various other groups such as amide, alcohol, halide, ether, amine, etc. The bromides are then converted into nitrites (24) using zinc cyanide and Pd(0) catalyst. 
The present invention encompasses the following Examples. Structural formulas follow. With regard to structural formulas, it is understood that oxygen and nitrogen atoms with available electrons have a hydrogen bound thereto, as indicated by compound name.